AICAR-Induced Activation of AMPK Inhibits TSH/SREBP-2/HMGCR Pathway in Liver

Our previous study found that thyroid-stimulating hormone promoted sterol regulatory element-binding protein-2 (SREBP-2) expression and suppressed AMP-activated protein kinase (AMPK) activity in the liver, but it was unclear whether there was a direct link between TSH, AMPK and SREBP-2. Here, we demonstrate that the 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced activation of AMPK directly inhibited the expression of SREBP-2 and its target genes HMGCR and HMGCS, which are key enzymes in cholesterol biosynthesis, and suppressed the TSH-stimulated up-regulation of SREBP-2 in HepG2 cells; similar results were obtained in TSH receptor knockout mice. Furthermore, AMPK, an evolutionally conserved serine/threonine kinase, phosphorylated threonine residues in the precursor and nuclear forms of SREBP-2, and TSH interacted with AMPK to influence SREBP-2 phosphorylation. These findings may represent a molecular mechanism by which AMPK ameliorates the hepatic steatosis and hypercholesterolemia associated with high TSH levels in patients with subclinical hypothyroidism (SCH).     

Decreased nuclear hormone receptor expression in the livers of mice in late pregnancy

During the third trimester of pregnancy, there is an increase in serum triglyceride and cholesterol levels. The mechanisms accounting for these changes in lipid metabolism during pregnancy are unknown. We hypothesized that, during pregnancy, the expression of nuclear hormone receptors involved in regulating lipid metabolism would decrease. In 19-day pregnant mice, serum triglyceride and non-HDL cholesterol levels were significantly increased, whereas total cholesterol was slightly decreased, because of a decrease in the HDL fraction. Peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and PPARgamma, liver X receptor (LXR)alpha and LXRbeta, farnesoid X receptor (FXR), and retinoid X receptor (RXR)alpha, RXRbeta, and RXRgamma mRNA levels were significantly decreased in the livers of 19-day pregnant mice. Additionally, the expressions of thyroid receptor (TR)alpha, pregnane X receptor, sterol regulatory element-binding proteins (SREBP)-1a, SREBP-1c, SREBP-2, and liver receptor homolog 1 were also decreased, whereas the expression of TRbeta, constitutive androstane receptor, and hepatic nuclear factor 4 showed no significant change. mRNA levels of the PPAR target genes carnitine-palmitoyl transferase 1alpha and acyl-CoA oxidase, the LXR target genes SREBP1c, ATP-binding cassettes G5 and G8, the FXR target gene SHP, and the TR target genes malic enzyme and Spot14 were all significantly decreased. Finally, the expressions of PPARgamma coactivator (PGC)-1alpha and PGC-1beta, known activators of a number of nuclear hormone receptors, were also significantly decreased. The decreases in expression of RXRs, PPARs, LXRs, FXR, TRs, SREBPs, and PGC-1s could contribute to the alterations in lipid metabolism during late pregnancy.     

Modulation of sterol regulatory element binding protein-2 in response to rapid follicle development in chickens

We investigated the expression profiles of sterol regulatory element binding proteins (SREBPs) and their related genes in chicken developing follicle membranes, from the small white follicle (SWF) stage to the Follicle 1 (F1) stage. Expression of SREBP-2 was significantly increased in the rapid stages of follicle development, however, no significant change in SREBP-1 mRNA expression was observed during follicle development. Immunoreactive SREBP-2 protein levels isolated from nuclear extracts in rapid growth stages, particularly in Follicle 2, were higher than those in SWF and small yellow follicle (SYF). In contrast, SREBP-1 immunoreactive protein levels were only slightly changed over all stage of follicle development. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR) mRNA levels significantly increased in the rapid stages of follicle development, suggesting that SREBP-2 controls the biosynthesis of cholesterol in follicles. LDL receptor and LDL receptor related protein 1 mRNA also tended to increase with follicular development, however, expression of LDL receptor relative with eight ligand binding repeats (LR8) was only slightly affected by SREBP-2. Liver X receptor α (LXR α) was expressed in chicken follicles; its expression patterns corresponded with SREBP-2 gene expression. These results suggest that SREBP-2, which might be regulated by LXRα, is involved in the rapid growth stages of follicle development in avian species.     

High density lipoprotein inhibits the activation of sterol regulatory element-binding protein-1 in cultured cells

A link between cellular uptake of high density lipoprotein (HDL) and regulation of sterol regulatory element-binding protein-1 (SREBP-1) was investigated in vitro. HDL decreased nuclear SREBP-1 levels as well as SREBP-1 target gene expression in HepG2 and HEK293 cells. However, HDL did not repress an exogenously expressed, constitutively active form of SREBP-1. HDL increased cellular cholesterol levels, and cellular cholesterol depletion by methyl-β-cyclodextrin abolished the effects of HDL. These results suggest that HDL inhibits the activation of SREBP-1 through a cholesterol-dependent mechanism, which may play an important role in regulating lipid synthetic pathways mediated by SREBP-1.     

The impact of severe LDL receptor mutations on SREBP-pathway regulation in homozygous familial hypercholesterolemia (FH)

Familial hypercholesterolemia (FH), Niemann-Pick disease type C (NPC) and Tangier disease (TD) are genetic inherited disorders with impaired processing of cholesterol, caused by mutations in genes that regulate cellular uptake, intracellular movement and transport of cholesterol. Various studies have shown a crucial regulatory role of the SREBP-pathway for cellular cholesterol homeostasis in these diseases. Since cholesterol is an essential structural component of cells, we assessed the impact of a severe FH causing LDLR mutation (FH p.W556R) on the SREBP pathway in primary FH fibroblasts. Primary FH fibroblasts derived from patients with the LDL receptor mutation p.W556R were used for gene expression experiments. Gene expression studies revealed increased expressions of SREBP regulated genes HMGCR, LDLR, SREBP-2, SREBP-1, SR-BI, INSIG-1, but interestingly not SCAP. In contrast expression of ABCA1, was strongly decreased in homozygous, but not in heterozygous p.W556R fibroblasts. Gene expression experiments with LDL receptor lacking primary FH fibroblasts, revealed that SR-BI and ABCA1 are important regulators for cholesterol acquisition in FH cells, consistent with findings in cells from NPC and TD patients.     

Cholesterol accumulation and diabetes in pancreatic beta-cell-specific SREBP-2 transgenic mice: a new model for lipotoxicity

To determine the role of cholesterol synthesis in pancreatic beta-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in beta-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in beta-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in beta-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of beta-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken together, these results indicate that activation of SREBP-2 in beta-cells caused severe diabetes by loss of beta-cell mass with accumulation of cholesterol, providing a new lipotoxic model and a potential link of disturbed cholesterol metabolism to impairment of beta-cell function.     

Increased cholesterol biosynthesis and hypercholesterolemia in mice overexpressing squalene synthase in the liver

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirus-mediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis.     

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.     

Effects of statins on the secretion of human serum albumin in cultured HepG2 cells

Statins reduce cholesterol biosynthesis by inhibiting HMG-CoA reductase and thereby lower total cholesterol and LDL cholesterol levels in serum, which in turn lower the incidence of cardiovascular disease (CVD). Statins are also known to modulate various cellular functions such as gene expression, cell proliferation, and programmed cell death through inhibition of downstream intermediates in cholesterol synthesis. In this study, we have investigated the possible effects of statins on the secretion of serum albumin from cultured HepG2 cells since high levels of serum albumin are associated with reduced risks for CVD and statins are effective in lowering the risk of CVD through other effects in addition to their effects on serum total cholesterol and LDL cholesterol levels, known as pleiotropic effects. Our results showed that simvastatin increased HSA secretion up to 32.3% compared to the control group. Among 3 statin analogs we tested, simvastatin exhibited the highest stimulatory effects on HSA secretion compared to the control group. Our study also showed that the increased HSA secretions from HepG2 cells by simvastatin treatments were due to the increased rate of HSA synthesis, not due to the reduced posttranslational degradation rate of HSA. Our finding suggests another added benefit of statins'' treatments in preventing CVD through stimulation of HSA biosynthesis.